Cross talk between oncogene and ant-oncogene via the Crk oncogene product

通过 Crk 癌基因产品实现癌基因和抗癌基因之间的交互

• 批准号: 10470064
• 负责人: MATSUDA Michiyuki
• 金额: $ 8.51万
• 依托单位: Osaka University (2001)Research Institute, International Medical Center of Japan (1998-2000)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10470064/
• 关键词: Crk; FRET; fluorescent resonance energy transfer; signal transduction; adaptor protein; Ras; Rap1; biotechnology; 上皮細胞増殖因子; リン酸化; Rap2; グアニンヌクレオチド交換因子; GTP水解促進酵素; C3G; 癌遺伝子; 癌抑制遺伝子

Crk oncogene product activates Ras and Rap1. Ras is a well-known oncogene, whereas Rap1 antagonize Ras-dependent oncogenesis. To understand the mechanism by which Crk activates both oncogene and anti-oncogene, we developed a FRET-based single-molecule probe that can monitor the activation of Crk in a living cell. Using this probe, we visualized rapid Crk activation in an EGF-stimulated cell. However, with the prototype probe, we could not determine where in the cell the activation of Crk starts, due to rapid diffusion of the probe. To overcome this flaw, we fused CAAX box of Ki-Ras protein to the probe, expecting the anchoring of probe to the plasma membrane. Indeed, by the use of this CAAX-fused probe, we found that Crk was activated from the peripheral region of the cell. Furthermore, we generated a recombinant adenovirus for the expression of the probe, which enabled us rapid and efficient expression of the probe in a variety of cells. Thus, the development of new probe for the Crk activity visualized the spatio temporal regulation of Crk oncogene products in living cells and promised us for the understanding of the role of Crk oncogene products in the regulation of cell growth and transformation.

Crk 癌基因产物激活 Ras 和 Rap1。 Ras 是一种众所周知的癌基因，而 Rap1 则拮抗 Ras 依赖性的肿瘤发生。为了了解 Crk 激活癌基因和抗癌基因的机制，我们开发了一种基于 FRET 的单分子探针，可以监测活细胞中 Crk 的激活。使用该探针，我们观察到 EGF 刺激的细胞中 Crk 的快速激活。然而，使用原型探针时，由于探针的快速扩散，我们无法确定 Crk 的激活在细胞中的哪个位置开始。为了克服这个缺陷，我们将 Ki-Ras 蛋白的 CAAX 盒融合到探针上，期望探针锚定在质膜上。事实上，通过使用这种 CAAX 融合探针，我们发现 Crk 从细胞的外围区域被激活。此外，我们生成了用于表达探针的重组腺病毒，这使得我们能够在多种细胞中快速有效地表达探针。因此，Crk活性新探针的开发使活细胞中Crk癌基因产物的时空调节可视化，并为我们了解Crk癌基因产物在细胞生长和转化调节中的作用提供了帮助。

项目成果

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Hasekawa H 等人：“DOCK180 是一种主要的 CRK 结合蛋白，在易位到膜上后会改变细胞形态。”

K.Kurakawa, et al.: "A Pair of Fluorescent Resonance Energy Transfer-based Probes for Tyrosine phosphorylation of the CrkII Adaptor Protein in Vivo."J. Boil Chem.. 276. 31305-31310 (2001)

K.Kurakawa 等人：“一对基于荧光共振能量转移的探针，用于体内 CrkII 衔接蛋白的酪氨酸磷酸化”。

Yamashita, S., N. Mochizuki, Y. Ohba, M. Tobiume, Y. Okada, H. Sawa, K. Nagashima, and <M. Matsuda>__________-: "CalDAG-GEFIII activation of Ras, R-Ras, and Rap1"J. Biol. Chem.. 275. 25488-25493 (2000)

山下，S.，N. Mochizuki，Y. Ohba，M. Tobiume，Y. Okada，H. Sawa，K. Nagashima，和 <M.

Dolfi F,et al.: "The adapter protein Crk regulates the JNK signaling pathway in response to multiple cellular stimuli." Proc Natl Acad Sci USA. 95. 15394-15399 (1998)

Dolfi F 等人：“接头蛋白 Crk 调节 JNK 信号通路以响应多种细胞刺激。”

Hashimoto Y,et al.: "Phosphorylation of CrkII adaptor protein at Tyrosine 221 by epidermal growth factor receptor." J Biol Chem. 273. 17186-17191 (1998)

Hashimoto Y 等人：“表皮生长因子受体对 CrkII 接头蛋白的酪氨酸 221 进行磷酸化。”